Abstract
Direct manipulation of bacterial chromosomes by recombination-based techniques has become increasingly important for both cognitive and applied research. Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic olignucleotides. We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated two component signaling system PhoPQ. The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.
Highlights
The generation of markerless deletions and mutations within a bacterial genome is of increasing importance for both cognitive and applied research
Techniques based on l Red-mediated recombination were previously shown to be very versatile for generating gene deletions in Escherichia coli [1], Salmonella enterica [2], Pseudomonas aeruginosa [3] as well as Shigella spp. [4] and Yersinia enterocolitica [5] or Y. pestis [6]
After confirming proper insertion of the resistance cassette by colony polymerase chain reaction (PCR), 80mer DNA fragments derived from oligonucleotides are electroporated in the mutant strain expressing the l Red recombinase system
Summary
The generation of markerless deletions and mutations within a bacterial genome is of increasing importance for both cognitive and applied research. Homologous recombination catalyzed by l Red is remarkably efficient using linear DNA with homology extensions of only 36 to 40 bp in length [1,8] With this technique, a chromosomal sequence of varying length is replaced by a selectable antibiotic resistance marker [1]. Integrating a tetracycline resistance cassette in the first step enabled us to select for successful recombinants after the second recombination using growth suppression of Tetr clones on Bochner-Maloy plates [9,10]. The exact mechanism how growth of tetracycline-resistant clones is inhibited on Bochner-Maloy plates is still unknown Since this selection procedure is not very stringent, it requires exact timing of incubation steps and results sometimes in high background making purification of positive clones difficult. Because of the statistical absence of natural I-SceI recognition sites within bacterial genomes, the enzyme was previously used as a tool for genetic engineering [13,14,15]
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